Methods and materials for reducing  venous neointimal hyperplasia of an arteriovenous fistula or graft

ABSTRACT

This document provides methods and materials involved in reducing venous neointimal hyperplasia (VNH) of an arteriovenous fistula (AVF) or graft. For example, methods and materials for using stem cells (e.g., mesenchymal stem cells), extracellular matrix material, or a combination of stem cells and extracellular matrix material to reduce VNH of AVFs or grafts are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/400,962, filed May 1, 2019, which is a divisional of U.S. patentapplication Ser. No. 15/097,070, filed Apr. 12, 2016 (now U.S. Pat. No.10,286,116), which claims the benefit of U.S. Provisional ApplicationSer. No. 62/166,241, filed May 26, 2015 and U.S. Provisional ApplicationNo. 62/147,762, filed Apr. 15, 2015. The disclosures of the priorapplications are considered part of (and are incorporated by referencein) the disclosure of this application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under HL098967 awardedby National Institutes of Health. The government has certain rights inthe invention.

BACKGROUND 1. Technical Field

This document relates to methods and materials involved in reducingvenous neointimal hyperplasia (VNH) of an arteriovenous fistula (AVF) orgraft. For example, this document provides methods and materials forusing stem cells (e.g., mesenchymal stem cells), extracellular matrixmaterial, or a combination of stem cells and extracellular matrixmaterial to reduce VNH of AVFs or grafts.

2. Background Information

In the United States, more than 350,000 patients with end-stage renaldisease (ESRD) are being treated using hemodialysis. The maintenance ofvascular access patency is essential for providing optimal hemodialysisin patients with ESRD. AVFs are the preferred manner for providingvascular access for hemodialysis. Only 60% of patients, however, have afunctional AVF after one year. The major cause for AVF failure is VNH,which leads to the development of stenosis and subsequent thrombosis(Roy-Chaudhury et al., Kidney International, 59:2325-2334 (2001)).

SUMMARY

This document provides methods and materials for reducing VNH of an AVFor graft. For example, this document provides methods and materials forusing stem cells (e.g., mesenchymal stem cells), extracellular matrixmaterial, or a combination of stem cells and extracellular matrixmaterial to reduce VNH of AVFs or grafts. As described herein, stemcells (e.g., adipose-derived mesenchymal stem cells) can be administeredto the adventitia of the outflow vein to reduce the development of VNHassociated with AVFs. As also described herein, extracellular matrixmaterial (e.g., an extracellular matrix scaffold such as CorMatrix™) canbe applied to (e.g., wrapped around) the adventitia of the outflow veinof AVFs to reduce the development of VNH associated with AVFs. Havingthe ability to reduce development of VNH of an AVF or graft using themethods and materials provided herein can allow clinicians and patientsto maintain the function of AVFs or grafts whether involved inhemodialysis or other types of grafting procedures.

The methods and materials provided herein can be used to reduce thedevelopment of VNH after peripheral and coronary artery bypass graftsurgery. In some cases, the methods and materials provided herein can beused in conjunction with angioplasty or stent placement. For example,stem cells (e.g., mesenchymal stem cells), extracellular matrixmaterial, or both can be delivered using an endovascular catheterconfigured to target the adventitia. In some cases, the methods andmaterials provided herein can be used with endovascular delivery to theendothelium with or without using angioplasty, stents, or nanoparticles.In some cases, stem cells (e.g., mesenchymal stem cells) can beadministered as described herein during angioplasty or stent placement.In some cases, extracellular matrix material can be applied to (e.g.,wrapped around) the adventitia during or after a peripheral or coronaryarterial bypass surgery.

In general, one aspect of this document features a method for reducingvenous neointimal hyperplasia formation of an arteriovenous fistula orgraft in a mammal. The method comprises, or consists essentially of,administering stem cells to an adventitia of a vein of the arteriovenousfistula or graft under conditions wherein venous neointimal hyperplasiaformation of the arteriovenous fistula or graft is reduced. The mammalcan be a human. The stem cells can be adipose-derived mesenchymal stemcells.

In another aspect, this document features a method for reducing venousneointimal hyperplasia formation of an arteriovenous fistula or graft ina mammal. The method comprises, or consists essentially of, applyingextracellular matrix material to an adventitia of a vein of thearteriovenous fistula or graft under conditions wherein venousneointimal hyperplasia formation of the arteriovenous fistula or graftis reduced. The mammal can be a human. The extracellular matrix materialcan be porcine extracellular matrix material. The extracellular matrixmaterial can be applied by wrapping the extracellular matrix materialaround the adventitia of the vein.

In another aspect, this document features a method for reducing venousneointimal hyperplasia formation of an arteriovenous fistula or graft ina mammal. The method comprises, or consists essentially of, (a)administering stem cells to an adventitia of a vein of the arteriovenousfistula or graft, and (b) applying extracellular matrix material to theadventitia, wherein venous neointimal hyperplasia formation of thearteriovenous fistula or graft is reduced. The mammal can be a human.The stem cells can be adipose-derived mesenchymal stem cells. Theextracellular matrix material can be porcine extracellular matrixmaterial. The extracellular matrix material can be applied by wrappingthe extracellular matrix material around the adventitia of the vein.

In another aspect, this document features a method for reducing venousneointimal hyperplasia formation of an arteriovenous fistula in amammal. The method comprises, or consists essentially of, implanting astent comprising extracellular matrix material into a blood vessel ofthe arteriovenous fistula under conditions wherein venous neointimalhyperplasia formation of the arteriovenous fistula is reduced. Themammal can be a human. The extracellular matrix material can be porcineextracellular matrix material. The extracellular matrix material can belocated between an inner wall of the blood vessel and an outer surfaceof the stent.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of AVF placement with MSC.

FIG. 2. Localization of human adipose derived mesenchymal stem cells(MSCs). 2.5×10⁵ MSCs stable transfected with GFP were injected into theadventitia of the outflow vein of AVF at the time of creation. GFPlabeled human adipose derived mesenchymal stem cells (MSCs) were presentat day 7 in MSC transplanted vessels (M) compared to outflow veinvessels removed from control animals (C) after AVF placement. Nuclei areblue. There are GFP positive cells (arrows) in the vessel wall of theoutflow vein at day 7. The lumen is marked with an *.

FIG. 3. Monocyte chemoattractant protein-1 (Mcp-1) gene expression atday 7 in MSC transplanted vessels (M) compared to outflow vein vesselsremoved from animals (C). There was a significant decrease in the meanMcp-1 gene expression in the M transplanted vessels when compared to Cgroup (P<0.05). Each bar shows the mean±SEM of 4-6 animals per group.Two-way ANOVA with Student t test was performed. Significant differencesamong M transplanted and C vessels are indicated by *P<0.05.

FIGS. 4A-4D. Histomorphometric analysis of mesenchymal stem celltransplanted vessels (M) compared to outflow vein vessels removed fromanimals with AVF only (C) at day 7 and 21 after placement. FIG. 4A is arepresentative section after Hematoxylin and eosin (H and E) staining inM transplanted or C vessels at day 7 after AVF placement. n isneointima, and m+a is media/adventitia. All are original magnification×40. Bar is 50-mM. * is lumen. FIG. 4B is semiquantitative analysisrevealing a significant increase in the average lumen vessel area of Mtransplanted vessels when compared to control C group for day 7 (P<0.05)and 21 (P<0.05). FIG. 4C is a semiquantitative analysis for the averagearea of the neointima/average area of the media/adventitia for bothgroups at day 7 and 21. By day 21, there was a significant decrease inthe average area of the neointima/average area of the media/adventitiain the M transplanted vessels when compared to the C group (P<0.05).FIG. 4D is a semiquantitative analysis for the average cell density inthe neointima for both groups at days 7 and 21. By day 7, the averagecell density of the neointima in the M transplanted vessels wassignificantly lower than the C group (P<0.0001) and remained lower byday 21 (P<0.05). Each bar represents mean±SEM of 4-6 animals per group.Two-way ANOVA followed by Student t-test with post hoc Bonferroni'scorrection was performed. Significant differences among M transplantedand C vessels are indicated by *P<0.05 or ##P<0.0001.

FIGS. 5A-5C. TdT-mediated dNTP nick end labeling (TUNEL) staining andKi-67 staining in murine AVF at day 7 and 21 after placement of AVF inoutflow vein alone (C) and MSC transplanted vessels (M). FIG. 5A (upperpanel) is the representative sections from TUNEL staining at outflowvein of the M transplanted and C vessels at day 7 and 21. Brown stainingnuclei were positive for TUNEL. Negative control is shown where therecombinant terminal deoxynucleotidyl transferase enzyme was omitted.All are original magnification ×40. Bar is 50-mM. * is lumen. FIG. 5B isthe semiquantitative analysis for TUNEL staining for M transplanted andC vessels at days 7 and 21. By day 7, the average density of cellsstaining positive for TUNEL (brown staining nuclei) at the outflow veinof M transplanted vessels was significantly higher than the C group(P<0.0001). By day 21, it remained significantly increased (P<0.05).FIG. 5A (lower panel) is representative sections after Ki-67 staining inoutflow vessels removed mesenchymal stem cell transplanted vessels (M)or arteriovenous fistula alone (C) at day 7 after AVF placement. Brownstaining nuclei are positive for Ki-67. IgG negative controls are shown.FIG. 5C is the semiquantitative analysis of Ki-67 staining at days 7 and21. By day 7, there was a significant reduction in the average Ki-67index in the M transplanted vessels when compared to C vessels(P<0.0001) and remained significantly lower by day 21 (P<0.05). Each barrepresents mean±SEM of 4-6 animals. Two-way ANOVA followed by Studentt-test with post hoc Bonferroni's correction was performed. Significantdifferences among M transplanted and C vessels are indicated by *P<0.05,**P<0.001, or ##P<0.0001.

FIGS. 6A-6C. Fibroblast specific protein-1 (FSP-1) and α-smooth musclecell actin (α-SMA) staining in murine AVF at day 7 and 21 afterplacement of AVF in outflow vein alone (C) and MSC transplanted vessels(M). FIG. 6A (upper panel) is the representative sections after FSP-1staining in the venous stenosis of the M transplanted and C vessels atdays 7 and 21. Brown staining cells are positive for FSP-1. IgG negativecontrols are shown. All are original magnification ×40. Bar is 50-mM. *is lumen. FIG. 6B is the semiquantitative analysis of FSP-1 staining forM transplanted and C vessels at days 7 and 21. By day 7, the averagedensity of cells staining positive for FSP-1 at the outflow vein of Mtransplanted vessels was significantly lower than the C group (P<0.001).By day 21, it remained significantly increased (P<0.01). FIG. 6A (lowerpanel) is the representative sections after α-SMA staining in the venousstenosis of the M transplanted and C vessels at days 7 and 21. Brownstaining cells are positive for α-SMA. FIG. 6C is the semiquantitativeanalysis of α-SMA staining for M transplanted and C vessels at days 7and 21. By day 21, the average density of cells staining positive forα-SMA at the outflow vein of M transplanted vessels was significantlylower than the C group (P<0.05). Each bar represents mean±SEM of 4-6animals per group. Two-way ANOVA followed by Student t-test with posthoc Bonferroni's correction was performed. Significant differences amongM transplanted and C vessels are indicated by *P<0.05, #P<0.01, or##P<0.0001.

FIGS. 7A-7C. HIF-1α and CD68 staining in murine AVF at day 7 and 21after placement in outflow vein alone (C) and MSC transplanted vessels(M). FIG. 7A (upper panel) is the representative sections after HIF-1αstaining in the venous stenosis of the M transplanted and C vessels atdays 7 and 21. Brown staining nuclei were positive for HIF-1α. IgGnegative controls are shown. All are original magnification ×40. Bar is50-mM. * is lumen. FIG. 7B is the semiquantitative analysis of HIF-1αstaining for M transplanted and C vessels at days 7 and 21. By day 7,the average density of cells staining positive for HIF-1α at the outflowvein of M transplanted vessels was significantly lower than the C group(P<0.0001). By day 21, it remained significantly increased (P<0.0001).FIG. 7A (lower panel) is the representative sections after CD68 stainingin the venous stenosis of the M transplanted and C vessels at days 7 and21. Brown staining cells are positive for CD68. FIG. 7C is thesemiquantitative analysis of CD68 staining for M transplanted and Cvessels at days 7 and 21. By day 7, the average density of cellsstaining positive for CD68 at the outflow vein of M transplanted vesselswas significantly lower than the C group (P<0.05). Each bar representsmean±SEM of 4-6 animals per group. Two-way ANOVA followed by Studentt-test with post hoc Bonferroni's correction was performed. Significantdifferences among M transplanted and C vessels is indicated by *P<0.05or ##P<0.0001.

FIGS. 8A-8D. Histomorphometric analysis in CorMatrix™ scaffold wrappedvessels (S) when compared to control AVF (C). FIG. 8A is arepresentative section after Hematoxylin and eosin (H and E) staining inscaffold (S) wrapped or control (C) vessels at day 7 after AVFplacement. n is neointima, and m+a is media/adventitia. All wereoriginal magnification ×40. Bar is 50-mM. * is lumen. FIG. 8B is asemi-quantitative analysis that shows a significant increase in theaverage lumen vessel area of S wrapped vessels when compared to controlC group for day 21 (P<0.0001). FIG. 8C is a semiquantitative analysisfor the average area of the neointima for both groups at day 7 and 21.By day 7, there was a significant decrease in the average area of theneointima of the S wrapped vessels when compared to the C group(P<0.05). FIG. 8D is a semi-quantitative analysis for the average celldensity in the neointima for both groups at days 7 and 21. By day 21,the average cell density of the neointima in the S wrapped vessels wassignificantly lower than the C group (P<0.0001). Each bar representsmean±SEM of 4-6 animals per group. Two-way ANOVA followed by Studentt-test with post hoc Bonferroni's correction was performed. Significantdifferences among S wrapped and C vessels is indicated by *P<0.05 or##P<0.0001.

FIGS. 9A-9C. TdT-mediated dNTP nick end labeling (TUNEL) and Ki-67staining in CorMatrix™ scaffold wrapped vessels (S) when compared tocontrol AVF (C). FIG. 9A (upper panel) is the representative sectionsfrom TUNEL staining at outflow vein of the S wrapped and C vessels atday 7 and 21. Brown staining nuclei are positive for TUNEL. Negativecontrol is shown where the recombinant terminal deoxynucleotidyltransferase enzyme was omitted. All are original magnification ×40. Baris 50-mM. * is lumen. FIG. 9B is the semiquantitative analysis for TUNELstaining for S wrapped and C vessels at days 7 and 21. By day 7, theaverage density of cells staining positive for TUNEL (brown stainingnuclei) at the outflow vein of S wrapped vessels was significantlyhigher than the C group (P<0.0001). By day 21, it remained significantlyincreased (P<0.0001). FIG. 9A (lower panel) is representative sectionsafter Ki-67 staining in outflow vessels removed S wrapped vessels orarteriovenous fistula alone (C) at day 7 after AVF placement. Brownstaining nuclei are positive for Ki-67. IgG negative controls are shown.FIG. 9C is the semiquantitative analysis of Ki-67 staining at days 7 and21. By day 7, there was a significant reduction in the average Ki-67index in the S wrapped vessels when compared to C vessels (P<0.01). Eachbar represents mean±SEM of 4-6 animals. Two-way ANOVA followed byStudent t-test with post hoc Bonferroni's correction was performed.Significant differences among S wrapped and C vessels are indicated by#P<0.01 or ##P<0.0001.

FIGS. 10A-10C. α-SMA and FSP-1 staining in CorMatrix™ scaffold wrappedvessels (S) when compared to control AVF (C). FIG. 10A (upper panel) isthe representative sections after α-SMA staining in the venous stenosisof the S wrapped and C vessels at days 7 and 21. Brown staining cellsare positive for α-SMA. IgG negative controls are shown. All areoriginal magnification ×40. Bar is 50-mM. * is lumen. FIG. 10B is thesemiquantitative analysis of α-SMA staining for S wrapped and C vesselsat days 7 and 21. By day 21, the average density of cells stainingpositive for α-SMA at the outflow vein of S wrapped vessels wassignificantly lower than the C group (P<0.01). FIG. 10A (lower panel) isthe representative sections after FSP-1 staining in the venous stenosisof the S wrapped and C vessels at days 7 and 21. Brown staining cellsare positive for FSP-1. FIG. 10C is the semiquantitative analysis ofFSP-1 staining for S wrapped and C vessels at days 7 and 21. By day 7,the average density of cells staining positive for FSP-1 at the outflowvein of S wrapped vessels was significantly lower than the C group(P<0.01). By day 21, it remained significantly increased (P<0.0001).Each bar represents mean±SEM of 4-6 animals per group. Two-way ANOVAfollowed by Student t-test with post hoc Bonferroni's correction wasperformed. Significant differences among S wrapped and C vessels areindicated by #P<0.01, or ##P<0.0001.

FIGS. 11A-11C. HIF-1α and CD68 staining in CorMatrix™ scaffold wrappedvessels (S) when compared to control AVF (C). FIG. 11A (upper panel) isthe representative sections after HIF-1α staining in the venous stenosisof the S wrapped and C vessels at days 7 and 21. Brown staining nucleiare positive for HIF-1α. IgG negative controls are shown. All areoriginal magnification ×40. Bar is 50-mM. * is lumen. FIG. 11B is thesemiquantitative analysis of HIF-1α staining for S wrapped and C vesselsat days 7 and 21. By day 7, the average density of cells stainingpositive for HIF-1α at the outflow vein of S wrapped vessels wassignificantly lower than the C group (P<0.0001). By day 21, it remainedsignificantly increased (P<0.0001). FIG. 11A (lower panel) is therepresentative sections after CD68 staining in the venous stenosis ofthe S wrapped and C vessels at days 7 and 21. Brown staining cells arepositive for CD68. FIG. 11C is the semiquantitative analysis of CD68staining for S wrapped and C vessels at days 7 and 21. By day 7, theaverage density of cells staining positive for CD68 at the outflow veinof S wrapped vessels was significantly lower than the C group (P<0.05).Each bar represents mean±SEM of 4-6 animals per group. Two-way ANOVAfollowed by Student t-test with post hoc Bonferroni's correction wasperformed. Significant differences among S wrapped and C vessels areindicated by *P<0.05 or ##P<0.0001.

FIG. 12. Serial PET images of ⁸⁹Zr distribution in mice afteradventitial delivery of ⁸⁹Zr-labeled MSCs or ⁸⁹Zr (HPO₄)₂. The anatomicreference skeleton images were formed by using the mouse atlasregistration system algorithm with information obtained from thestationary top-view planar x-ray projector and side-view optical camera.SUV=standardized uptake value.

DETAILED DESCRIPTION

This document provides methods and materials for reducing VNH formationof an AVF or graft. For example, this document provides methods andmaterials for using stem cells to reduce VNH formation of AVFs orgrafts. As described herein, administering stem cells to the adventitiaof a vein of an AVF or graft of a mammal can reduce VNH formation ascompared to the level of VNH formation observed in a control mammal notreceiving the stem cells. In addition, applying an extracellular matrixmaterial to the adventitia of a vein of an AVF or graft (e.g., wrappingan extracellular matrix scaffold around the adventitia) can reduce VNHformation as compared to the level of VNH formation observed in acontrol mammal not receiving the extracellular matrix material. In somecases, both administering stem cells to the adventitia and applying anextracellular matrix material to the adventitia can be used to reduceVNH formation of AVFs or grafts as compared to the level of VNHformation observed in a control mammal not receiving the stem cells orthe extracellular matrix material.

Any appropriate mammal having an AVF or graft can be treated asdescribed herein. For example, humans, monkeys, dogs, cats, horses,cows, pigs, sheep, rats, and mice having an AVF or graft can be receivestem cells and/or extracellular matrix material as described herein toreduce VNH formation of the AVF or graft.

Examples of stem cells that can be used as described herein include,without limitation, mesenchymal stem cells such as adipose-derivedmesenchymal stem cells, bone marrow-derived mesenchymal stem cells,umbilical cord tissue-derived mesenchymal stem cells, and placentalderived mesenchymal stem cells. Examples of extracellular matrixmaterial that can be used as described herein include, withoutlimitation, porcine extracellular matrix material such as CorMatrix™(material manufactured by Cook Biotech (West Lafayette, Ind.) forCorMatrix Cardiovascular, Inc. (Atlanta, Ga.)) and extracellular matrixmaterial derived from plants, mice, rats, rabbits, cows, dogs, monkey,sheep, or baboons.

Any appropriate number of stem cells (e.g., adipose-derived mesenchymalstem cells) can be administered to the adventitia of a vein of an AVF orgraft. For example, between about 1×10⁶ and 1×10¹² stem cells (e.g.,adipose-derived mesenchymal stem cells) can be injected into theadventitia of a vein of an AVF or graft. In some cases, a singleadministration can be performed to reduce VNH formation of AVFs orgrafts. In some cases, multiple administrations can be performed toreduce VNH formation of AVFs or grafts. For example, stem cells (e.g.,adipose-derived mesenchymal stem cells) can be injected two, three,four, five, six, or more times to reduce VNH formation of AVFs orgrafts.

Any appropriate method can be used to apply extracellular matrixmaterial to the adventitia of a vein of an AVF or graft. For example,extracellular matrix material in the form of a strip or sheet can bewrapped partially (e.g., at least about 25 percent around, at leastabout half way around, or at least about 75 percent around) orcompletely around the adventitia of a vein of an AVF or graft.

In some cases, the ability of stem cells, extracellular matrix material,or both to reduce VNH formation of AVFs or grafts can be monitored. Anymethod can be used to determine whether or not VNH formation is reduced.For example, ultrasound, intravascular ultrasound, angiogram, computedtomographic analysis, or magnetic resonance angiography can be used toassess possible VNH formation.

In some cases, stem cells, extracellular matrix material, or both can beapplied to a stent. For example, a stent can be coated withextracellular matrix material and implanted into a blood vessel of amammal. In some cases, extracellular matrix material can be applied to astent in a manner such that drugs (e.g., anti-vascular endothelialgrowth factor-A or calcitriol), viruses (e.g., engineered lentivirusesor adenoviruses), small molecule inhibitors, and/or anti-miRNAs aredelivered to a mammal. For example, a stent can be coated withextracellular matrix material that contains a drug (e.g., anti-vascularendothelial growth factor-A or calcitriol), virus (e.g., an engineeredlentivirus or adenovirus), a small molecule inhibitor, and/or ananti-miRNA. Such a coated stent can be implanted into a blood vessel ofa mammal to deliver the drug, virus, a small molecule inhibitor, and/oran anti-miRNA to the mammal. In some cases, the outer surface of a stentcan include extracellular matrix material. In such cases, upondeployment of the stent into a blood vessel, the extracellular matrixmaterial can be located between an inner wall of the blood vessel and anouter surface of the stent. In some cases, all the surfaces of animplanted stent can be coated with extracellular matrix material.

In some cases, a stent coated with stem cells, extracellular matrixmaterial, or both as described herein can be implanted into a bloodvessel of an AVF. For example, stent coated with an extracellular matrixmaterial (e.g., an extracellular matrix material containing a drug,virus, a small molecule inhibitor, and/or an anti-miRNA) can beimplanted into a blood vessel of an AVF to reduce VNH.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Human Adipose Derived Mesenchymal Stem CellTransplantation to the Adventitia of the Outflow Vein Attenuates VNHAssociated with AVFs Experimental Animals

Animals were housed at 22° C. temperature, 41% relative humidity, and12-/12-hour light/dark cycles. Animals were allowed access to water andfood ad libitum. CD1-Foxn1nu mice weighting 20-25 g and agesapproximately 6-8 weeks were purchased from the Charles RiverLaboratories (Wilmington, Mass.). These animals lack a thymus, areunable to produce T cells, and are therefore immunodeficient which isideal for xenograft research. Anesthesia was achieved withintraperitoneal injection of a mixture of ketamine hydrochloride(0.1-0.2 mg/g) and xylazine (0.02 mg/g). Arteriovenous fistula (AVF)between right carotid artery to the ipsilateral jugular vein was createdas described elsewhere (Yang et al., J. Vasc. Interv. Radiol.,20:946-950 (2009)). 2.5×10⁵ MSC cells stably labeled with GFP in 5-μL ofmedia were injected into the adventitia of the outflow vein at the timeof AVF creation in the MSC group. Animals were sacrificed at day 7following AVF placement for real time polymerase chain reaction(qRT-PCR) and histomorphometric analyses and at day 21 forhistomorphometric analysis only.

Human MSCs Preparation

Human MSCs from healthy donors were obtained from the Human CellularTherapy Laboratory. These cells were characterized with respect tosurface markers and described elsewhere (Crespo-Diaz et al., CellTransplant, 20:797-811 (2011)). Briefly, they are CD73 (+), CD90 (+),CD105 (+), CD44 (+), and HLA-ABC (+).

GFP Transfection

MSCs were transfected with GFP lentivirus. MSCs were grown in mediacontaining the GFP lentivirus overnight. The media was changed tocomplete growth media the next day, and cells were checked forfluorescence after 48 hours. Once fluorescence was confirmed, the cellswere cultured in complete media containing 1 μg/mL puromycin. Cellscontaining the plasmid were expanded in complete growth media.

⁸⁹Zr Labeling and In Vivo Tracking of Stem Cells

Noninvasive PET imaging was used to evaluate the biodistribution of MSCsdelivered to the adventitia outside the AVF in CD1-Foxn1nu mice. Forthis, MSCs were labeled with a biostable radiolabeling synthon,⁸⁹Zr-desferrioxamine-N-chlorosuccinimide, as described elsewhere (Bansalet al., EJNMMI Res., 5:19 (2015)). After delivery of 2×10⁵ ⁸⁹Zr-labeledMSCs (at a radioactivity concentration of approximately 0.55 MBq per 10⁶cells) into the adventitia, the ⁸⁹Zr-labeled MSCs were tracked for 3weeks by using a small-animal PET/radiography system (Genesys4; SofieBioSystems, Culver City, Calif.). In the control group, 0.28 MBq of ⁸⁹Zr(HPO₄)₂ was delivered into the adventitia. PET images were normalized tounits of standardized uptake value, which was calculated as follows:tissue radioactivity concentration/(injected dose/body weight in grams).

Immunohistochemistry

After fixation with formalin and processing, the samples were embeddedin paraffin. Histological sectioning began at the outflow vein segment.Routinely, 80 to 120, 5-μm sections were obtained, and the cuff used tomake the anastomosis could be visualized. Every 25-μm, 2-4 sections werestained with Hematoxylin and eosin, Ki-67, α-SMA, HIF-1α, or CD68, orTUNEL was performed on the paraffin-embedded sections from the outflowvein. The EnVision (DAKO, Carpinteria, Calif.) method was used with aheat-induced antigen retrieval step (Misra et al., Kidney International,68:2890-2900 (2005)). The following antibodies were used: mousemonoclonal antibody Ki-67 (DAKO, 1:400) or rabbit polyclonal antibody tomouse for CD68, α-SMA, or HIF-1α (Abcam, 1:600). IgG antibody stainingwas performed to serve as controls.

TUNEL Staining

TUNEL staining was performed on paraffin-embedded sections from theoutflow vein of MSC with scaffold (e.g., CorMatrix™) as specified by themanufacturer (DeadEnd Colorimetric tunnel assay system, G7360, Promega).Negative control is shown where the recombinant terminaldeoxynucleotidyl transferase enzyme was omitted.

Morphometry and Image Analysis

Five-μm paraffin embedded sections were immunostained and quantified asdescribed elsewhere (Yang et al., Kidney International, 85:289-306(2014)).

RNA Isolation

The outflow vein was isolated and stored in RNA stabilizing reagent(Qiagen, Gaithersburg, Md.) as per the manufactures guidelines. Toisolate the RNA, the specimens were homogenized, and total RNA from thesamples was isolated using RNeasy mini kit (Qiagen) (Yang et al., J.Vasc. Interv. Radiol., 20:946-950 (2009)).

Real Time Polymerase Chain Reaction (qRT-PCR) Analysis

Expression for the gene of interest was determined using qRT-PCRanalysis as described elsewhere (Yang et al., J. Vasc. Interv. Radiol.,20:946-950 (2009)). Primers used are shown in Table 1.

TABLE 1  Mouse primers used SEQ ID Gene Sequence NO: MCP-15′-GGAGAGCTACAAGAGGATCAC-3′ (sense) 15′-TGATCTCATTTGGTTCCGATCC-3′ (antisense) 2 iNOS5′-TTGCTCATGACATCGACCAG-3′ (sense) 35′-ACATCAAAGGTCTCACAGGC-3′ (antisense) 4 Arg-15′-CCCAGATGTACCAGGATTCTC-3′ (sense) 55′-AGCTTGTCTACTTCAGTCATGG-3′ (antisense) 6 18S5′-GTTCCGACCATAAACGATGCC-3′ (sense) 75′-TGGTGGTGCCCTTCCGTCAAT-3′ (antisense) 8

Statistical Methods

Data were expressed as mean±SEM. Multiple comparisons were performedwith two-way ANOVA followed by Student t-test with post hoc Bonferroni'scorrection. Significant difference from control value was indicated by*P<0.05, #P<0.01, **P<0.001, or ##P<0.0001. JMP version 9 (SAS InstituteInc., Cary, N.C.) was used for statistical analyses.

Results Surgical Outcomes

Forty-seven male B6.Cg-Foxn1nu/J mice underwent the placement of carotidartery to jugular vein fistula as described elsewhere (Janardhanan etal., Kidney International, August:338-352 (2013); Yang et al., KidneyInternational, 85:289-306 (2014); and Yang et al., J. Vasc. Interv.Radiol., 20:946-950 (2009)). Eleven mice died after celltransplantation. 2×10⁵ MSCs labeled with GFP were injected into theadventitia of the outflow vein at the time of AVF creation (FIG. 1).Animals were sacrificed at day 7 for either histomorphometric or qRT-PCRanalyses for each of the following groups: AVF only (C, n=6) and MSC (M,n=6). Another group of animals were sacrificed at day 21 after fistulaplacement for histomorphometric and immunohistochemical analyses for thefollowing groups: AVF only (C, n=6) and MSC (M, n=6).

Localization of MSCs After Adventitial Delivery of to the Outflow Veinof AVF

In order to assess the spatial and temporal localization of MSCs to theadventitia of the outflow vein, MSCs were stably transfected with GFP inorder to track them. Confocal microscopy of the outflow vein afteradventitial transplantation of MSC was performed at different times.This demonstrated that GFP positive cells from the M transplantedvessels (blue positive cells, FIG. 2, arrow head) were present at day 7.However, by day 21, there was no co-localization of the GFP signal fromeither group.

PET images of mice after adventitial delivery of ⁸⁹Zr-labeled MSCsrevealed that more than 90% of administered ⁸⁹Zr radioactivity wasretained at the delivery site on day 4 (FIG. 12). Adventitial retentionof ⁸⁹Zr radioactivity cleared slowly from day 4 to day 21, losingapproximately 20% over this period (FIG. 12). Most ⁸⁹Zr radioactivitythat was cleared from the adventitia appeared to translocate to bones.This result confirmed the results obtained using confocal microscopywith GFP-labeled cells on day 7. PET imaging of ⁸⁹Zr-labeled MSCsallowed tracking of cells beyond 7 days, which was not possible withGFP-labeled cells. The retention of most of the delivered stem cells atthe delivery site on day 21 demonstrates that the effect was longer thanwhat was visualized using GFP labeling. In the case of the controlgroup, in which ⁸⁹Zr (HPO₄)₂ was administered, a biodistribution similarto that of ⁸⁹Zr-labeled MSCs was seen, with most of the radioactivity(approximately 80%) retained at the delivery site and the restredistributing to bones.

Adventitial Transplantation of MSC Reduces Gene Expression of Mcp-1

Studies demonstrated that MSCs exert their anti-inflammatory effectthrough a reduction in gene expression of Mcp-1 (Wise et al., Am. J.Physiol. Renal Physiol., 306:F1222-1235 (2014)). The gene expression ofMcp-1 was assessed by performing qRT-PCR analysis at day 7 (FIG. 3A). Mtransplanted vessels were compared to control AVFs alone. The averagegene expression of Mcp-1 at outflow vein of M transplanted vessels wassignificantly lower than the C group (average reduction: 70%, P<0.05).

Adventitial transplantation of MSC to the outflow vein reduced theaverage neointima area/media+adventitia area and cell density in theneointima while increasing the average lumen vessel area at days 7 and21.

The vascular remodeling of the outflow vein in the M transplantedvessels and C vessels at day 7 and 21 was determined usinghistomorphometric analysis as described elsewhere (Janardhanan et al.,Kidney International, August:338-352 (2013); and Yang et al., KidneyInternational, 85:289-306 (2014)). By examining the Hematoxylin andeosin stained sections, one was able to differentiate between theneointima (n) and media/adventitia (m+a, FIG. 4A). The average lumenvessel area was determined at day 7 and a significant increase in theoutflow vein removed from M transplanted vessels versus C group (averageincrease: 176%, P<0.001) was observed. By day 21, it remainedsignificantly increased in the M transplanted vessels when compared to Cgroup (average increase: 415%, P<0.0001). The average of the neointimaarea/media+adventitia area also was determined. By day 21, there was asignificant decrease in the neointima area/media+adventitia area in theoutflow vein removed from the M transplanted vessels when compared tothe C group (average reduction: 77%, P<0.05, FIG. 4C).

Neointimal hyperplasia is characterized by cell proliferation, celldifferentiation, and extra cellular matrix deposition (Roy-Chaudhury etal., Kidney International, 59:2325-2334 (2001); Rekhter et al.,Arterioscler. Thromb., 13:609-617 (1993); and Swedberg et al.,Circulation, 80:1726-1736 (1989)). The cell density was determined toassess if reduction of neointimal area was caused by change in celldensity. By day 7, the average cell density of the neointima in the Mtreated vessels was significantly lower than the C group (averagereduction: 83%, P<0.0001, FIG. 4C). By day 21, it remained lower in theM transplanted vessels when compared to the C group (average reduction:83%, P<0.0001, FIG. 4C).

Adventitial Transplantation of MSC to the Outflow Vein Increases TUNELStaining

The decrease in cell density might be due to an increase in apoptosis(Shay-Salit et al., Proc. Natl. Acad. Sci. USA, 99:9462-9467 (2002)).Apoptosis was evaluated by using TUNEL staining (FIG. 5A, upper panel).By day 7, the average density of cells staining positive for TUNEL(brown staining nuclei) at the outflow vein of M transplanted vesselswas significantly increased compared to the C group (average increase:180%, P<0.0001). By day 21, it remained higher in the M transplantedvessels compared to the C group (average increase: 427%, P<0.0001).These results demonstrate that M transplanted vessels have increasedTUNEL activity indicating cellular apoptosis when compared to C vessels.

Adventitial Transplantation of MSC to the Outflow Vein Reduces CellularProliferation at the Outflow Vein

Ki-67 staining was used to assess a possible association betweendecreased cellular density and a reduction in cellular proliferation.Brown staining nuclei were positive for Ki-67 (FIG. 5A, lower panel). Byseven days after fistula placement, there was a significant reduction inthe average Ki-67 index in the M transplanted vessels when compared to Cgroup (average reduction: 81%, P<0.001, FIG. 5C). By day 21, it remainedsignificantly lower in the M transplanted vessels when compared to Cvessels (average reduction: 60%, P<0.05).

Adventitial Transplantation of MSC to the Outflow Reduces α-SMA andFSP-1 Staining

Fibroblast specific protein-1 (FSP-1) was used as a fibroblast marker(FIG. 6A, upper panel). Smooth muscle deposition was assessed usingα-SMA staining (FIG. 6A, lower panel). Other studies implicatedfibroblast to myofibroblast (α-SMA) differentiation resulting in VNH(Misra et al., Kidney International, 68:2890-2900 (2005); and Wang etal., European Renal Association; 23:525-533 (2008)). By day 7, asignificant decrease in the average FSP-1 staining was observed in the Mtransplanted vessels when compared to C group (average reduction: 65%,P<0.001, FIG. 6B). By day 21, it remained significantly lower in the Mtransplanted vessels when compared to C group (average reduction: 42%P<0.01). By day 21, the average α-SMA staining was significantly lowerin the M transplanted vessels when compared to C group (averagereduction: 27%; P<0.05, FIG. 6C). Overall these results indicate that atday 7 there is a reduction in FSP-1 staining followed by a decrease inα-SMA staining by day 21 in M transplanted vessels when compared to Cvessels.

Adventitial Transplantation of MSC to the Outflow is Associated with aReduction in HIF-1α Staining

Other studies demonstrated increased HIF-1α expression in animal modelsof hemodialysis graft failure and in clinical specimens from patientswith hemodialysis vascular access failure (Misra et al., J. Vasc.Interv. Radiol., 21:1255-1261 (2010); and Misra et al., J. Vasc. Interv.Radiol., 19:252-259 (2008)). HIF-1α staining was quantified to assesswhether MSC transplantation had an effect on the expression of HIF-1α atthe outflow vein of AVF. Brown staining nuclei were positive for HIF-1α(FIG. 7A, upper panel). By day 7, there was a significant reduction inthe average density of HIF-1α staining M transplanted vessels whencompared to C vessels (average reduction: 67%, P<0.0001). By day 21, itremained significantly lower in the M treated vessels when compared to Cvessels (average reduction: 62%, P<0.0001). Overall these resultsindicate that there is decreased expression in HIF-1α in M transplantedvessels when compared to C treated vessels.

Adventitial Transplantation of MSC to the Outflow is Associated with aReduction in CD68 Staining

Other studies demonstrated increased CD68 expression (a marker formacrophages) in animal models of hemodialysis graft failure and inclinical specimens from patients with hemodialysis vascular accessfailure (Misra et al., J. Vasc. Interv. Radiol., 21:1255-1261 (2010);and Misra et al., J. Vasc. Interv. Radiol., 19:252-259 (2008)). CD68staining was quantified to assess whether MSC transplantation had aneffect on the expression of macrophage at the outflow vein of AVF. Cellsstaining brown in the cytoplasm were positive for CD68 (FIG. 7A, lowerpanel). By day 7, there was a significant reduction in the averagedensity of CD68 staining in the M treated vessels when compared to Cvessels (average reduction: 51%, P<0.05, FIG. 7C). Overall, there is asignificant decrease in CD68 staining in the M transplanted vessels whencompared to controls.

These results demonstrate that adventitial transplantation of humanadipose derived MSCs to the outflow vein of AVF in a murine modelreduces VNH. This is mediated by a significant decrease in the geneexpression of Mcp-1 in the outflow vein transplanted with MSCs comparedto controls at day 7. There was a significant increase in average TUNELstaining with a decrease in proliferation. In addition, there was asignificant decrease in the FSP-1, CD68, and α-SMA staining accompaniedwith a decrease in average HIF-1α staining.

Example 2 Wrapping an Extracellular Matrix Scaffold Around theAdventitia of the Outflow Vein of AVFs Attenuates VNH ExperimentalAnimals

Animals were housed at 22° C. temperature, 41% relative humidity, and12-/12-hour light/dark cycles. Animals were allowed access to water andfood ad libitum. CD1-Foxn1nu mice weighting 20-25 g and agesapproximately 6-8 weeks were purchased from the Charles RiverLaboratories (Wilmington, Mass.). These animals lack a thymus, areunable to produce T cells, and are therefore immunodeficient which isideal for xenograft research. Anesthesia was achieved withintraperitoneal injection of a mixture of ketamine hydrochloride(0.1-0.2 mg/g) and xylazine (0.02 mg/g). AVF between right carotidartery to the ipsilateral jugular vein was created as describedelsewhere (Yang et al., J. Vasc. Interv. Radiol., 20:946-950 (2009)).1×4 mm CorMatrix™ scaffolds were wrapped around the outflow vein andsutured using 8-0 nylon to secure the scaffold to the outflow vein atthe time of AVF creation (FIG. 1). Animals were sacrificed at day 7 and21 for histomorphometric analyses for AVF only (C, n=6) or scaffoldalone (S, n=6).

CorMatrix™ Scaffold Wrapped Around the Adventitia of the Outflow Vein ofthe AVF

The scaffold (CorMatrix™) material was created by Cook Biotech (WestLafayette, Ind.) for CorMatrix Cardiovascular, Inc. (Atlanta, Ga.) andis composed of porcine small-intestine submucosa (SIS). Physically, theSIS was about 155-μm thick with pore sizes up to 50 μm when hydrated.The scaffolds were cut to 1×4-mm.

Immunohistochemistry

After fixation with formalin and processing, the samples were embeddedin paraffin. Histological sectioning began at the outflow vein segment.80 to 120, 5-μm sections were obtained, and the cuff used to make theanastomosis could be visualized. Every 0.1 mm, 2-4 sections were stainedwith Hematoxylin and eosin, Ki-67, α-SMA, or HIF-1α, or TUNEL wasperformed on paraffin-embedded sections from the outflow vein. TheEnVision (DAKO, Carpinteria, Calif.) method was used with a heat-inducedantigen retrieval step (Misra et al., Kidney International, 68:2890-2900(2005)). The following antibodies were used: mouse monoclonal antibodyKi-67 (DAKO, 1:400) or rabbit polyclonal antibody to mouse for α-SMA andHIF-1α (Abcam, 1:600). IgG antibody staining was performed to serve ascontrols.

TUNEL Staining

TUNEL staining was performed on paraffin-embedded sections from theoutflow vein of Scaffold treated vessels and AVF only as specified bythe manufacturer (DeadEnd Colorimetric tunnel assay system, G7360,Promega). Negative control is shown where the recombinant terminaldeoxynucleotidyl transferase enzyme was omitted.

Morphometry and Image Analysis

Five-μm paraffin embedded sections were immunostained and quantified asdescribed elsewhere (Yang et al., Kidney International, 85:289-306(2014)).

Statistical Methods

Data were expressed as mean±SEM. Multiple comparisons were performedwith two-way ANOVA followed by Student t-test with post hoc Bonferroni'scorrection. Significant differences from control value were indicated by*P<0.05 or #P<0.01. JMP version 9 (SAS Institute Inc., Cary, N.C.) wasused for statistical analyses.

Results Surgical Outcomes

Twenty-four male B6.Cg-Foxn1nu/J mice underwent the placement of carotidartery to jugular vein fistula as described elsewhere (Janardhanan etal., Kidney International, August:338-352 (2013); Yang et al., KidneyInternational, 85:289-306 (2014); and Yang et al., J. Vasc. Interv.Radiol., 20:946-950 (2009)). Animals were sacrificed at day 7 and 21 forhistomorphometric analyses for AVF only (C, n=6) or scaffold alone (S,n=6).

CorMatrix™ Wrapped Outflow Vein has Reduced Average Neointima Area andCell Density with Increased Average Lumen Vessel Area

The vascular remodeling of the outflow vein in the different groups atday 7 and 21 was determined using histomorphometric analysis asdescribed elsewhere (Janardhanan et al., Kidney International,August:338-352 (2013); and Yang et al., Kidney International, 85:289-306(2014)). Examining the Hematoxylin and eosin stained sections allowedfor the differentiation between the neointima (n) and media/adventitia(m+a, FIG. 8A). By day 21, the average lumen vessel area wassignificantly increased in the outflow vein removed from the S treatedvessels when compared to the C alone (average increase: 1800%,P<0.0001). The average area of the neointima was determined. By day 7,there was a significant decrease in the average area of the neointima inthe outflow vein removed from the S treated vessels when compared to theC group (average reduction: 77%, P<0.05, FIG. 8B). By day 21, there wasno difference between the two groups. Finally, the cell density wasdetermined to assess if the reduction in neointimal area was caused bychange in cell density. By day 21, the average cell density of theneointima in the S treated vessels was significantly lower than the Cgroup (average reduction: 83%, P<0.001, FIG. 8C).

CorMatrix™ Wrapped Outflow Vein has Increased TUNEL Staining whenCompared to Control Vessels

It is possible that the decrease in cell density also was due to anincrease in apoptosis. Apoptosis was evaluated by using TUNEL staining(FIG. 9A, upper panel). By day 7, the average density of cells stainingpositive for TUNEL (brown staining nuclei) at the outflow vein of Streated group was significantly higher than the C treated group (averageincrease: 38200%, P<0.0001, FIG. 9B). By day 21, the average density ofcells staining positive for TUNEL at the outflow vein of S treated groupwas significantly higher than the C treated group (average increase:516%, P<0.0001).

CorMatrix™ Wrapped Outflow Vein has Reduced Cellular Proliferation whenCompared to Control Vessels

Because the cellular density was decreased, Ki-67 staining was performedto determine if this was associated with a reduction in cellularproliferation. Brown staining nuclei were positive for Ki-67 (FIG. 9A,lower panel). By 7 days after fistula placement, there was a significantreduction in the average Ki-67 staining in the S treated vessels whencompared to C treated vessels (average reduction: 39%, P<0.0001, FIG.9C). By day 21, there was no difference between the two groups.

CorMatrix™ Wrapped Outflow Vein has Reduced α-SMA and FSP-1 Stainingwhen Compared to Control Vessels

Smooth muscle deposition was assessed using α-SMA staining (FIG. 10A,upper panel). There was no difference between the two groups at day 7,however, by day 21, the average α-SMA staining was significantly lowerin the S treated vessels when compared to C group (average reduction:55% P<0.05, FIG. 10B). FSP-1, a marker for fibroblasts, was used toidentify the presence of such cells (FIG. 10A, lower panel). Otherstudies have implicated fibroblast to myofibroblast (α-SMA)differentiation as resulting in VNH (Misra et al., Kidney International,68:2890-2900 (2005); and Wang et al., European Renal Association;23:525-533 (2008)). By day 7, a significant decrease in the averageFSP-1 staining was observed in the S treated vessels when compared to Cgroup (average reduction: 55%, P<0.01, FIG. 10C). By day 21, the averageFSP-1 staining remained significantly lower in the S treated vesselswhen compared to C group (average reduction: 55% P<0.001). Overall,these results indicate that at day 7 there was a reduction in FSP-1staining followed by a decrease in α-SMA staining by day 21 in S treatedvessels when compared to C vessels.

CorMatrix™ Wrapped Outflow Vein has Reduced HIF-1α Staining whenCompared to Control Vessels

Other studies demonstrated increased HIF-1α expression in animal modelsof hemodialysis graft failure and in clinical specimens from patientswith hemodialysis vascular access failure (Misra et al., J. Vasc.Interv. Radiol., 21:1255-1261 (2010); and Misra et al., J. Vasc. Interv.Radiol., 19:252-259 (2008)). HIF-1α staining was quantified to assesswhether S treated vessels had decreased expression of HIF-1α whencompared to controls. Brown staining nuclei were positive for HIF-1α(FIG. 11A, upper panel). By day 7, there was significant reduction inthe average density of HIF-1α staining in the S treated vessels whencompared to C vessels (average reduction: 71%, P<0.0001, FIG. 11B). Byday 21, the average density of HIF-1α staining remained significantlylower in the S treated vessels when compared to C vessels (averagereduction: 69%, P<0.0001).

CorMatrix™ Wrapped Outflow Vein has Reduced CD68 Staining when Comparedto Control Vessels

Other studies demonstrated increased CD68 expression (a marker formacrophages) in animal models of hemodialysis graft failure and inclinical specimens from patients with hemodialysis vascular accessfailure (Misra et al., J. Vasc. Interv. Radiol., 21:1255-1261 (2010);and Misra et al., J. Vasc. Interv. Radiol., 19:252-259 (2008)). CD68staining was quantified to assess whether the S treated vessels had aneffect on the expression of macrophage at the outflow vein of AVF. Cellsstaining brown in the cytoplasm were positive for CD68 (FIG. 11A, lowerpanel). By day 7, there was significant reduction in the average densityof CD68 staining in the S treated vessels when compared to C vessels(average reduction: 71%, P<0.0001, FIG. 11C) that remained significantlydecreased by day 21 (average reduction: 63%, P<0.05). Overall, theseresults indicate that S treated vessels when compared to C vessels havedecreased CD68 (+) staining.

These results demonstrate that extracellular matrix scaffolds (e.g.,CorMatrix™) can be used to wrap vessels in a manner that reduces VNH.This is accompanied by a significant increase in TUNEL staining and adecrease in proliferation. In addition, there is a significant decreasein the FSP-1, CD68, and α-SMA staining accompanied with a decrease inaverage HIF-1α staining.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for reducing venous neointimalhyperplasia formation of an arteriovenous fistula or graft in a mammal,wherein said method comprises administering stem cells to an adventitiaof a vein of said arteriovenous fistula or graft under conditionswherein venous neointimal hyperplasia formation of said arteriovenousfistula or graft is reduced.
 2. The method of claim 1, wherein saidmammal is a human.
 3. The method of claim 1, wherein said stem cells areadipose-derived mesenchymal stem cells.
 4. The method of claim 1,wherein administering said stem cells is during an angioplastyprocedure.
 5. The method of claim 1, wherein administering said stemcells is during a stent placement procedure.